Non-glycosylated transferrin expressed in monocots

ABSTRACT

Disclosed are compositions and methods of making non-glycosylated transferrin protein in transgenic monocot plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/102,966, filed 6 May 2011 and published as US 20120088729; andpursuant to 35 U.S.C. §119 (e), to the filing date of U.S. ProvisionalPatent Application Ser. No. 61/332,733 filed 7 May 2010, the disclosuresof each of which are herein incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This work was supported in part with government support under NIH grantGM086916 from the National Institute of General Medical Sciences. TheUnited States government may have rights to certain aspects of thedisclosure.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The instant application includes a sequence listing in the form of atext file created 6 May 2011, named “506658035US00SeqList.txt” (63488bytes) as well as submitted in the form of a paper copy, each of whichis incorporated into the application by reference in its entirety.

INTRODUCTION

The present teachings relate to monocot seeds and seed compositionscontaining levels of transferrin protein between 3-40% or higher of thetotal protein weight of the soluble protein extractable from the seed,and methods of producing high levels of non-glycosylated transferrinprotein in transgenic monocots, for use in making a serum-free cellculture medium, as well as animal, in particular human, therapeuticcompositions.

BACKGROUND

Iron is an element used by eukaryotic organisms and most microorganismsas a cofactor of numerous proteins or enzymes for respiration, DNAsynthesis, and many other critical metabolic processes (Baker, et al.,Proc Natl Acad Sci USA 100: 3579-3583 (2003)). Cellular iron deficiencycan arrest cell proliferation and even cause cell death, whereas theexcessive iron will be toxic to cells by reacting with oxygen via theFenton reaction to produce highly reactive hydroxyl radicals that causeoxidative damage to cells (Baker, et al., Proc Natl Acad Sci USA 100:3579-3583 (2003); Hentze, M. U., et al., Cell 117: 285-97(2004)). Toovercome the dual challenges of iron deficiency and overload, a familyof iron carrier glycoproteins collectively called transferrins hasevolved in nearly all organisms to tightly control cellular iron uptake,storage, and transport to maintain cellular iron homeostasis (Williams,J., Trends Biochem. Soc. 7: 394-397 (1982)). The transferrin proteinfamily includes several homologous glycoproteins generally having amolecular weight of approximately 80 kDa and an ability to bind iron,and is divided into four subsets: (1) serum transferrins (TF) which havea role in iron transport in the body; (2) lactoferrins (LF) found inmammalian extracellular secretions such as milk, tears, pancreatic fluidand other bodily secretions of mammals; (3) melanotransferrins (mTF)which is present on the surface of melanocytes and in liver andintestinal epithelium; and (4) ovotransferrins (oTF) found in bird andreptile oviduct secretions and egg white. While all members of thetransferrin protein family can bind iron to control free iron level,human serum transferrin provides both a means of transporting iron fromthe sites of absorption and storage to the sites of utilization, as wellas protection against the damaging effects of iron-catalyzed freeradicals. To date, only TF has been proven to be able to transport ironto cells (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583 (2003)).

One exemplary TF is a single-chain glycoprotein of 679 amino acidresidues including 38 cysteine residues which are all disulfide bonded.TF consists of two homologous halves, each comprising about 340 aminoacid residues and sharing about 40% sequence identity (Baker, et al.,Proc Natl Acad Sci USA 100: 3579-3583 (2003); Hirose, Biosci.Biotechnol. Biochem. 64:1328-1336 (2000); J. Wally, et al., Biometals20: 249-62 (2007)). The two homologous halves are shown by X-raycrystallography to fold into two distinct globular lobes called N- andC-terminal lobes (Baker, et al., Proc Natl Acad Sci USA 100: 3579-3583(2003); Hirose, Biosci. Biotechnol. Biochem. 64:1328-1336 (2000)). Eachlobe comprises two dissimilar domains (N1 and N2 in the N-lobe; C1 andC2 in the C-lobe) separated by a deep cleft, where the iron binding siteis located. The iron-binding ligands in each lobe are identical, whichinvolves the side chains of an aspartic acid, two tyrosines, a histidineand two oxygen molecules from a synergistic carbonate anion (Baker, etal., Proc Natl Acad Sci USA 100: 3579-3583 (2003); Hentze, M. U., etal., Cell 117: 285-97(2004); Hirose, Biosci. Biotechnol. Biochem.64:1328-1336 (2000); J. Wally, et al., Biometals 20: 249-62 (2007);Q.-Y. He, et al., “Molecular aspects of release of iron fromtransferrin,” in: D. M. Templeton, (Ed.), Molecular and Cellular IronTransport, CRC Press, 2002, pp. 95-124).

The cellular iron uptake and transport is normally driven by a TF/TFreceptor (TFR)-mediated endocytotic process (Baker, et al., Proc NatlAcad Sci USA 100: 3579-3583 (2003)). When TF is free of iron (apo-TF),both its N- and C-lobes adopt an open conformation through keeping twodomains in each lobe well separated for easy access of the ferric iron.At the extracellular pH of 7.4, the apo-TF binds one (monoferric TF) ortwo iron molecules (diferric TF or holo-TF) by the coordination ofiron-binding ligands. The diferric TF then binds to TFR on the cellsurface in a way that the TF C-lobe binds laterally at the helicaldomain of dimeric TFR while the TF N-lobe is sandwiched between the TFRectodomain and the cell membrane (Cheng, et al., Cell 116: 565-76(2004); Cheng, et al., J. Struct. Biol. 152: 204-210 (2005)). ThisTF-TFR complex is then endocytosed into the early endosome, where theacidic environment (pH 5.5) triggers the conformational change of TF-TFRand the subsequent release of iron from TF by first protonating anddissociating the synergistic anion followed by protonating ironbinding-related His and/or Tyr ligands (Baker, et al., Proc Natl AcadSci USA 100: 3579-3583 (2003); Q.-Y. He, et al., “Molecular aspects ofrelease of iron from transferrin,” in: D. M. Templeton, (Ed.), Molecularand Cellular Iron Transport, CRC Press, 2002, pp. 95-124). Finally, theapo-TF-TFR complex is recycled to the cell surface, where the neutralextracellular pH will dissociate the complex and release the TF forre-use.

The TF-TFR complex-mediated endocytosis pathway for iron transport isnot only biologically significant for maintaining cellular ironhomeostasis, but also has important pharmaceutical applications. TF isalso an important ingredient of serum-free cell culture media due to itsrole in regulating cellular iron uptake, transport, and utilization incultured cells. TF in serum-free cell culture medium ensures irondelivery to propagating cells for sustained growth in mammalian culturefor the production of therapeutic proteins and vaccines (Barnes, et al.,Cell 22: 649-55 (1980); Laskey, et al., Exp. Cell Res. 176: 87-95(1988); Mortellaro, et al., Biopharm. International 20 (Supp) 30-37(2007); Sharath, et al., J Lab Clin Med 103: 739-48 (1984)).

In addition, TF has also been actively pursued as a drug-deliveryvehicle. As a drug carrier, TF increases a drug's therapeutic index viaits unique transferrin receptor-mediated endocytosis pathway, as well asits added advantages of being biodegradable, nontoxic, andnonimmunogenic (Qian, et al., Med. Res. Rev. 22: 225-50 (2002); Qian, etal. , Pharmacol. Rev. 54: 561-87 (2002); Soni, et al., American Journalof Drug Delivery 3: 155-70 (2005)). TF not only can deliver anti-cancerdrugs to primary proliferating malignant cells where the TF isabundantly expressed (Qian, et al. , Pharmacol. Rev. 54: 561-87 (2002)),but also can deliver drugs to the brain by crossing the blood-brainbarrier (BBB), which is a major barrier for administrating sufficientdrugs to reach the central nervous system (CNS) (Qian, et al., Med. Res.Rev. 22: 225-50 (2002); Soni, et al., American Journal of Drug Delivery3: 155-70 (2005); Pardridge, Discov. Med. 6:139-43(2006)). TF can alsobe exploited for oral delivery of protein-based therapeutics (Bai, etal., Proc. Natl. Acad. Sci. U.S.A. 102: 7292-6 (2005); Widera, et al.,Adv. Drug Deliv. Rev. 55:1439-66(2003)), as TF is resistant toproteolytic degradation and TFR is abundantly expressed in humangastrointestinal (GI) epithelium (Bai, et al., Proc. Natl. Acad. Sci.U.S.A. 102: 7292-6 (2005); Banerjee, et al., Gastroenterology 91: 861-9(1986)).

With the increasing concerns over the risk of transmission of infectiouspathogenic agents from the use of human or animal plasma-derived TFs inboth cell culture and drug delivery applications, recombinanttransferrin (rTF) is preferred to native TF (Keenan, et al.,Cytotechnology 51: 29-37(2006)). Recombinant human TF (rhTF) has longbeen pursued in a variety of expression systems (MacGillivray, et al.,“Transferrins” in: D. M. Templeton, (Ed.), Molecular and cellular irontransport, Marcel Dekker, New York, 2002, pp. 41-70), but proves to bechallenging largely due to hTF's complicated structural characteristicsas described above. The commonly used E. coli system for production ofrecombinant proteins has proved to be impractical for producing rhTF, asthe expressed rhTF protein remains in insoluble inclusion bodies and theyield of functionally active rhTF after renaturation is very limited(Hoefkens, et al., Int. J. Biochem. Cell Biol. 28: 975-82 (1996)).Although both the insect cell (baculovirus) (Ali, et al., Biochem. J.319 (Pt 1):191-5 (1996)) and mammalian cell (MacGillivray, et al.,“Transferrins” in: D. M. Templeton, (Ed.), Molecular and cellular irontransport, Marcel Dekker, New York, 2002, pp. 41-70) expression systemshave been shown to be able to express the bioactive rhTF, neither ofthem express at high enough levels to provide enough quantity to be afeasible source of commercial production, as well as being costprohibitive.

It is shown herein that when transferrin is expressed in bacterial,yeast, mammalian cells, and insect cell expression systems, theexpressed native transferrin protein bears a glycosylation patterncharacteristic of the host organism, i.e., animal cell-expressedtransferrin has an animal-type glycosylation pattern, andyeast-expressed transferrin has a yeast-type glycosylation pattern. Itis desirable to produce a biologically active transferrin protein thatis non-glycosylated for therapeutic use, to avoid possible allergic orimmunological reactivity. Recently, bioactive rhTF was expressed inSaccharomyces cerevisiae using a mutated transferrin gene in which twoof its N-linked glycosylation sites have been knocked out, and this rhTFbecame commercially available. (Sargent, et al., BioMetals (2006)19:513-519). However, this yeast-derived rhTF, still remains veryexpensive to produce (Millipore, Billerica, Mass.). To address theproblems of the shortage and the high cost of producing rhTF, as well asto meet a previously unmet need for producing high levels of annon-glycosylated human transferrin, alternative expression systems aredesirable.

With the advancement of plant molecular biology in general and theimprovement of plant transformation techniques in particular, planthosts have become a powerful system to produce recombinant proteinscost-effectively and on a large scale (Daniell, et al., Trends PlantSci. 6: 219-26 (2001); Lienard, et al., Biotechnol. Annu. Rev. 13:115-47 (2007); Twyman, et al., Expert Opin. Emerg. Drugs 10: 185-218(2005); Huang, et al., “ExpressTec: high level expression ofbiopharmaceuticals in cereal grains” in: K. J, (Ed.), ModernBiopharmaceuticals, Wiley VCH, 2005, pp. 931-47).

None of the aforementioned patents or publications discloses theproduction of non-glycosylated native transferrin protein in monocotseeds in high yield. It is desirable to provide for the production ofnon-glycosylated native transferrin protein in high yield free fromcontaminating source agents in order to provide a sufficient supply oftransferrin in serum-free cell culture medium as well as in therapeuticcompositions for the patient population with conditions treatable byadministration of transferrin protein.

SUMMARY

Due to the high risk of contamination with blood-borne pathogens fromthe use of human- or animal plasma-derived transferrin, it isadvantageous to produce recombinant transferrin from an alternativesource, such as a crop plant, for use as a substitute for native human-or animal plasma-derived transferrin. Production of transferrin proteinsin plants mitigates any possible contamination of the transferrinprotein fraction by human or animal viruses and other disease causativeagents found in human or animal plasma product fractions. In one aspect,the present disclosure provides expression of recombinant humantransferrin (rhTF) in monocots, for example rice (Oryza sativa L.)grains, at high levels of expression, e.g., 1% seed dry weight (10g/kg). The recombinant human transferrin was extracted with salinebuffers and then purified by a one-step anion exchange chromatographicprocess to greater than 95% purity. The rice-derived recombinant humantransferrin was biochemically and functionally characterized, and shownto be not only biochemically similar to the native human transferrin,but also functionally the same as native transferrin in terms ofreversible iron binding and promoting cell growth and productivity.Specifically, the expressed rhTF was shown to be non-N-glycosylated byMALDI and PNGase F enzyme digestion analyses although the entire aminoacid sequence of rhTF including its N-glycosylation sites had not beengenetically modified to remove N-linked glycosylation sites. Thismonocot-derived rhTF was proved to be not only biochemically similar tothe native hTF, but also functionally equivalent to the native hTF.Specifically, the monocot-derived rhTF reversibly bound iron andpromoted cell growth and productivity. The ease of extraction andpurification of recombinant hTF protein makes the present disclosure aviable system for commercial production of rhTF at high levels and lowcost. Thus, the monocot-derived recombinant human transferrin describedherein provides a safe and low cost alternative to human or animalplasma-derived transferrin for use in cell culture-basedbiopharmaceutical production of protein therapeutics and vaccines.

In one aspect, the disclosure provides a method of producing arecombinant non-glycosylated transferrin protein in monocot plant seeds,comprising the steps of:

(a) transforming a monocot plant cell with a chimeric gene comprising

-   -   (i) a promoter from the gene of a seed maturation-specific        monocot plant storage protein,    -   (ii) a first DNA sequence, operably linked to said promoter,        encoding a monocot plant seed-specific signal sequence capable        of targeting a polypeptide linked thereto to a monocot plant        seed endosperm cell, and    -   (iii) a second DNA sequence, linked in translation frame with        the first DNA sequence, encoding a natural transferrin protein,        wherein the first DNA sequence and the second DNA sequence        together encode a fusion protein comprising an N-terminal signal        sequence and the tranferrin protein;

(b) growing monocot plant from the transformed monocot plant cell for atime sufficient to produce seeds containing the transferrin protein; and

(c) harvesting the seeds from the plant, wherein the transferrin proteinconstitutes at least 0.1% seed weight of the harvested seeds.

In some embodiments, the transgenic monocot plant may further comprise anucleic acid that encodes at least one transcription factor selectedfrom the group consisting of O2 (encoded by the sequence set forth asSEQ ID NO: 20), PBF (encoded by the sequence set forth as SEQ ID NO: 21)and Reb (encoded by the sequence set forth as SEQ ID NO: 22).

The disclosure also provides a monocot plant seed-derived composition,selected from whole-seed food composition, a flour composition, anextract composition and a malt composition, prepared from the harvestedseeds obtained by the disclosed method. In certain embodiments, thetransferrin protein constitutes at least 1.0% of the dry weight theseed-derived composition.

The disclosure further provides a monocot seed-derived compositioncomprising an non-glycosylated transferrin protein, and at least onepharmaceutically acceptable excipient or nutrient, wherein thenon-glycosylated transferrin protein is produced in a monocot plantcontaining a nucleic acid sequence encoding the transferrin protein andis extracted from seed harvested from the monocot plant. The excipientor nutrient is from a heterologous source other than the monocot plant.The formulation can be used for parenteral, enteric, inhalation,intranasal or topical delivery.

A serum-free cell culture medium comprising an extract of monocot seedexpressing non-glycosylated transferrin protein and a method of makingthe serum-free cell culture medium are provided.

These and other objects and features of the claimed subject matter willbecome more fully apparent when the following detailed description isread in conjunction with the accompanying drawings and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a diagram of an exemplary construct for high levelexpression of transferrin in monocots.

FIG. 2 provides an immuno dot-blot expression analysis of transgenicrice seeds expressing hTF;

FIGS. 3A and 3B illustrate SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot analyses, respectively, of rhTF expressed inrice grain.

FIGS. 4A and 4B illustrate SDS-PAGE and immunoblot analyses,respectively, of tissue specific expression of rhTF in rice plant roots,stems, leaves, leaf sheaths, anthers with pollens, grain husks, pistils,immature seeds, and mature seeds.

FIG. 5 presents an SDS-PAGE analysis of different fractions uponpurification of rice-derived rhTF protein extracts.

FIG. 6 presents a MALDI mass spectrum molecular weight analysis ofpurified rice-derived rhTF.

FIG. 7 presents a glycosylation state analysis by PNGase F treatment ofrice-derived rhTF.

FIG. 8 presents an isoelectic focusing gel analysis of rice-derivedrhTF.

FIG. 9 provides a RP-HPLC comparison of rice-derived rhTF and native hTF(“nhTF”).

FIGS. 10A-D provide an analysis of iron-binding properties ofrice-derived rhTF.

FIGS. 11A-C presents an analysis of the effect of rhTF on cell growthand antibody production.

DETAILED DESCRIPTION

Several embodiments of the present disclosure are described in detailhereinafter. These embodiments may take many different forms and shouldnot be construed as limited to those embodiments explicitly set forthherein. Rather, these embodiments are provided so that this disclosurewill be thorough and complete, and will fully convey the scope of thepresent disclosure to those skilled in the art.

7.1 DEFINITIONS

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a “monocot plant” can mean, for example, a singlemonocot plant, such as a rice plant, or it can mean two or more of thesame or different species of monocot plants.

As used herein, the following terms are intended to have the followingmeanings:

The term “stably transformed” with reference to a plant cell means theplant cell has a non-native (heterologous) nucleic acid sequenceintegrated into its genome which is maintained through two or moregenerations.

“Chimeric gene” or “heterologous nucleic acid construct,” as definedherein refers to a construct which has been introduced into a host andmay include parts of different genes of exogenous or autologous origin,including regulatory elements. A chimeric gene construct for plant/seedtransformation is typically composed of a transcriptional regulatoryregion (promoter) operably linked to a heterologous protein codingsequence, or, in a selectable marker heterologous nucleic acidconstruct, to a selectable marker gene encoding a protein conferringantibiotic resistance to transformed plant cells. A typical chimericgene of the present disclosure, includes a transcriptional regulatoryregion inducible during seed development, a protein coding sequence, anda terminator sequence. A chimeric gene construct may also include asecond DNA sequence encoding a signal peptide if secretion of the targetprotein is desired.

The term “gene” means the segment of DNA involved in producing apolypeptide chain, which may or may not include regions preceding andfollowing the coding region, e.g. 5′ untranslated (5′ UTR) or “leader”sequences and 3′ UTR or “trailer” sequences, as well as interveningsequences (introns) between individual coding segments (exons).

The term “sequence identity” means nucleic acid or amino acid sequenceidentity in two or more aligned sequences, aligned using a sequencealignment program.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at (ncbi.nlm.gov/BLAST/). See, also,Altschul, S. F. et al., 1990 and Altschul, S. F. et al., 1997.

The term “% homology” is used interchangeably herein with the term “%identity” and refers to the level of nucleic acid or amino acid sequenceidentity between two or more aligned sequences, when aligned using asequence alignment program. For example, 70% homology means the samething as 70% sequence identity determined by a defined algorithm, andaccordingly a homologue of a given sequence has greater than 70%sequence identity over a length of the given sequence. Exemplary levelsof sequence identity include, but are not limited to 70%, 75% 80%, 85%,90% or 95% or more sequence identity to a given sequence, e.g., thecoding sequence for transferrin, as described herein.

Sequence searches are typically carried out using the BLASTN programwhen evaluating a given nucleic acid sequence relative to nucleic acidsequences in the GenBank DNA Sequences and other public databases. TheBLASTX program is preferred for searching nucleic acid sequences whichhave been translated in all reading frames against amino acid sequencesin the GenBank Protein Sequences and other public databases. Both BLASTNand BLASTX are run using default parameters of an open gap penalty of11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62matrix. (See ncbi.nlm.gov/BLAST/. See, also, Altschul, S. F. et al.,1990 and Altschul, S. F. et al., 1997).

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-W program in MacVector version 6.5, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

A nucleic acid sequence is considered to be “selectively hybridizable”to a reference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe. Forexample, “maximum stringency” typically occurs at about Tm-5° C. (5°below the Tm of the probe); “high stringency” at about 5-10° below theTm; “intermediate stringency” at about 10-20° below the Tm of the probe;and “low stringency” at about 20-25° below the Tm. Functionally, maximumstringency conditions may be used to identify sequences having strictidentity or near-strict identity with the hybridization probe; whilehigh stringency conditions are used to identify sequences having about80% or more sequence identity with the probe.

Moderate and high stringency hybridization conditions are well known inthe art (see, for example, Sambrook et al, 1989, Chapters 9 and 11, andin Ausubel et al., 1993, expressly incorporated by reference herein). Anexample of high stringency conditions includes hybridization at about42° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDS and 100μg/ml denatured carrier DNA followed by washing two times in 2×SSC and0.5% SDS at room temperature and two additional times in 0.1×SSC and0.5% SDS at 42° C.

“Heterologous DNA” refers to DNA which has been introduced into plantcells from another source, or which can be from a plant source,including the same plant source, but which is under the control of apromoter that does not normally regulate expression of the heterologousDNA.

“Heterologous protein” is a protein encoded by a heterologous DNA.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

A plant cell, tissue, organ, or plant into which a heterologous nucleicacid construct comprising the coding sequence for an anti-microbialprotein or peptide has been introduced is considered transformed,transfected, or transgenic. A transgenic or transformed cell or plantalso includes progeny of the cell or plant and progeny produced from abreeding program employing such a transgenic plant as a parent in across and exhibiting an altered phenotype resulting from the presence ofthe coding sequence for an anti-microbial protein. Hence, a plant of thepresent disclosure will include any plant which has a cell containingintroduced nucleic acid sequences, regardless of whether the sequencewas introduced into the plant directly through transformation means orintroduced by generational transfer from a progenitor cell whichoriginally received the construct by direct transformation.

The term “transgenic plant” refers to a plant that has incorporatedexogenous nucleic acid sequences, i.e., nucleic acid sequences which arenot present in the native (“untransformed”) plant or plant cell. Thus aplant having within its cells a heterologous polynucleotide is referredto herein as a “transgenic plant.” The heterologous polynucleotide canbe either stably integrated into the genome, or can beextra-chromosomal. The polynucleotide of the present disclosure may bestably integrated into the genome such that the polynucleotide is passedon to successive generations. The term “transgenic” as used herein doesnot encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.“Transgenic” is used herein to include any cell, cell line, callus,tissue, plant part or plant, the genotype of which has been altered bythe presence of heterologous nucleic acids including those transgenicsinitially so altered as well as those created by sexual crosses orasexual reproduction of the initial transgenics.

The terms “transformed,” “stably transformed” or “transgenic” withreference to a plant cell means the plant cell has a non-native(heterologous) nucleic acid sequence integrated into its genome which ismaintained through two or more generations.

The term “expression” with respect to a protein or peptide refers to theprocess by which the protein or peptide is produced based on the nucleicacid sequence of a gene. The process includes both transcription andtranslation. The term “expression” may also be used with respect to thegeneration of RNA from a DNA sequence.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection,” or “transformation” or“transduction” and includes the incorporation of a nucleic acid sequenceinto a eukaryotic or prokaryotic cell where the nucleic acid sequencemay be incorporated into the genome of the cell (for example,chromosome, plasmid, plastid, or mitochondrial DNA), converted into anautonomous replicon, or transiently expressed (for example, transfectedmRNA).

By “host cell” is meant a cell containing a vector and supporting thereplication and/or transcription and/or expression of the heterologousnucleic acid sequence.

A “plant cell” refers to any cell derived from a plant, includingundifferentiated tissue (e.g., callus) as well as plant seeds, pollen,propagules, embryos, suspension cultures, meristematic regions, leaves,roots, shoots, gametophytes, sporophytes and microspores.

The term “mature plant” refers to a fully differentiated plant.

The term “seed product” includes, but is not limited to, seed fractionssuch as de-hulled whole seed, a flour composition (seed that has beende-hulled by milling and ground into a powder) a seed extractcomposition, in some embodiments, a protein extract (where the proteinfraction of the flour has been separated from the carbohydratefraction), a malt composition (including malt extract or malt syrup)and/or a purified protein fraction derived from the transgenic grain.

The term “biological activity” refers to any biological activitytypically attributed to that protein by those of skill in the art.

The term “non-nutritional” refers to a pharmaceutically acceptableexcipient which does not as its primary effect provide nutrition to therecipient. The excipient may provide one of the following services to anenterically delivered formulation, including acting as a carrier for atherapeutic protein, protecting the protein from acids in the digestivetract, providing a time-release of the active ingredients beingdelivered, or otherwise providing a useful quality to the formulation inorder to administer to the patient the transferrin protein.

“Monocot seed components” refers to carbohydrate, protein, and lipidcomponents extractable from monocot seeds, typically mature monocotseeds.

“Seed maturation” refers to the period starting with fertilization inwhich metabolizable reserves, e.g., sugars, oligosaccharides, starch,phenolics, amino acids, and proteins, are deposited, with and withoutvacuole targeting, to various tissues in the seed (grain), e.g.,endosperm, testa, aleurone layer, and scutellar epithelium, leading tograin enlargement, grain filling, and ending with grain desiccation.

“Maturation-specific protein promoter” refers to a promoter exhibitingsubstantially upregulated activity (greater than 25%) during seedmaturation.

A “signal sequence” is an N- or C-terminal polypeptide sequence which iseffective to localize the peptide or protein to which it is attached toa selected intracellular or extracellular region. In some embodiments,the signal sequence targets the attached peptide or protein to alocation such as an endosperm cell, in certain embodiments, anendosperm-cell organelle, such as an intracellular vacuole or otherprotein storage body, chloroplast, mitochondria, or endoplasmicreticulum, or extracellular space, following secretion from the hostcell.

“Transferrin” can refer to a transferrin protein or protein-encodingsequence from an animal, such as a mammal, including a human. Exemplaryamino acid sequences for mammalian transferrins are disclosed herein asthe mature human transferrin protein Swiss-Prot accession number P02787,(identified herein as SEQ ID NO: 3); murine transferrin protein GenBankaccession AAL34533.1 (identified herein as SEQ ID NO: 24); rattransferrin protein GenBank accession BAA07458.1 (identified herein asSEQ ID NO: 25); porcine transferrin protein GenBank accession CAQ34904.1(identified herein as SEQ ID NO: 26); and macaque transferrin proteinGenBank accession ACB11584.1 (identified herein as SEQ ID NO: 27).

“Non-glycosylated” or “unglycosylated” means without observable N-linkedglycosylation, within the limits of detection by isoelectric focusing,PNGase F digestion and/or MALDI analysis. These terms make no referenceto or implications about the O-linked glycosylation status of a protein.

“Native transferrin” means transferrin protein that is not produced froma mutated recombinant gene.

“Plant-derived” means that the source of the ingredient is a plant.

“Dry weight percent” or “% dry weight” or “percent seed dry weight”means, for example, a protein-yield of grams transferrin per kilogram ofdry seeds. For example, 1% seed dry weight of rice-expressed transferrinmeans that 1 kilogram of rice grains yields 10 grams of transferrinprotein.

“Total protein” and “total soluble protein” are used interchangeably,unless otherwise specified. Thus, unless otherwise noted, any givenweight of total protein measured should be interpreted by the skilledartisan to mean total soluble protein. Further, a value given in μg/mgTSP to the corresponding value given in % TSP. As an example, 1 μg/1 mgTSP is equivalent to 1 μg per 1000 μg TSP (or 0.001 μg/μg TSP) which,expressed as a percentage of TSP in μg weight, would be 0.1% TSPmeasured in μg. For example, 30.3 μg/mg total (soluble) protein. Thistranslates to 0.0303 μg per μg TSP, which, stated as a percentage,equals 3.03% TSP.

Units can also be expressed as μg per grain of monocot seed. This weightcan be correlated with the percentage of total soluble protein, giventhat the average weight of a seed/grain and how much of this weight isrepresented by the TSP are matters readily known to skilled artisans.For example, the 1000 grain weight of rice is, on average, approximately20-25 grams, which translates to 20-25 mg (or 20,000-25,000 μg) per ricegrain. As one example, a transgenic rice plant may typically yield 190μg total soluble protein per grain which is roughly equivalent to 0.8%grain weight (190 μg divided by 25,000 μg=0.0076 which is rounded up to0.8%).

As is known in the art, “endosperm” or “endosperm tissue” is a seedstorage tissue found in mature seeds.

The terms “crude extract,” “partially-purified” or “substantiallyunpurified” means that a composition made from the transgenic monocotseed is not subjected to significant purification steps, such aschromatographic protein purification and fractionation steps.

1.2 DETAILED DESCRIPTION

In some embodiments, the host cell is a monocot plant cell, such as, forexample, a monocot endosperm cell. Other host cells may be used assecondary hosts, including bacterial, yeast, insect, amphibian ormammalian cells, to move DNA to a desired plant host cell.

The polynucleotides of the disclosure may be in the form of RNA or inthe form of DNA, and include messenger RNA, synthetic RNA and DNA, cDNA,and genomic DNA The DNA may be double-stranded or single-stranded, andif single-stranded may be the coding strand or the non-coding(antisense, complementary) strand.

Expression vectors for use in the present disclosure are chimericnucleic acid constructs (or expression vectors or cassettes), designedfor operation in plants, with associated upstream and downstreamsequences.

In general, expression vectors can include the following operably linkedcomponents that constitute a chimeric gene: a promoter from the gene ofa maturation-specific monocot plant storage protein, a first DNAsequence, operably linked to the promoter, encoding a monocot plantseed-specific signal sequence (such as an N-terminal leader sequence ora C-terminal trailer sequence) capable of targeting a polypeptide linkedthereto to an endosperm cell, in some embodiments an endosperm-cellorganelle, such as a protein storage body, and a second DNA sequence,linked in translation frame with the first DNA sequence, encoding atransferrin protein. The signal sequence may be cleaved from thetransferrin protein in the plant cell.

An exemplary DNA sequence encoding native human transferrin is set forthas SEQ ID NO: 1. An exemplary codon-optimized DNA sequence encodinghuman transferrin is set forth as SEQ ID NO: 2.

The chimeric gene, in turn, is typically placed in a suitableplant-transformation vector having (i) companion sequences upstreamand/or downstream of the chimeric gene which are of plasmid or viralorigin and provide necessary characteristics to the vector to permit thevector to move DNA from bacteria to the desired plant host; (ii) aselectable marker sequence; and (iii) a transcriptional terminationregion generally at the opposite end of the vector from thetranscription initiation regulatory region.

Numerous types of appropriate expression vectors, and suitableregulatory sequences are known in the art for a variety of plant hostcells. The promoter region is chosen to be regulated in a mannerallowing for induction under seed-maturation conditions. In one aspect,the expression construct includes a promoter which exhibits specificallyupregulated activity during seed maturation. Promoters are typicallyderived from cereals such as rice, barley, wheat, oat, rye, corn,millet, triticale or sorghum. Examples of such promoters include thematuration-specific promoter region associated with one of the followingmaturation-specific monocot plant storage proteins: rice glutelins,oryzins, and prolamines, barley hordeins, wheat gliadins and glutelins,maize zeins and glutelins, oat glutelins, and sorghum kafirins, milletpennisetins, and rye secalins. Exemplary regulatory regions from thesegenes are exemplified by SEQ ID NOS: 4-12. Some promoters suitable forexpression in maturing seeds include the barley endosperm-specificB1-hordein promoter, GluB-2 promoter, Bx7 promoter, Gt3 promoter, GluB-1promoter and Rp-6 promoter, particularly if these promoters are used inconjunction with transcription factors.

“Alpha-amylase” as used herein refers to an enzyme which principallybreaks starch into dextrins. “Beta-amylase” as used herein refers to anenzyme which converts start and dextrins into maltose. An exemplarycoding sequence of the rice alpha-amylase (RAmy3D) gene is set forth inGenBank accession M59351.1 (identified herein as SEQ ID NO: 28). SeeHuang, et al., Nucleic Acids Res. 18 (23), 7007-7014 (1990).

Of particular interest is the expression of the nucleic acid encoding atransferrin protein from a promoter that is preferentially expressed inplant seed tissue. Examples of such promoter sequences include thosesequences derived from sequences encoding plant storage protein genes orfrom genes involved in fatty acid biosynthesis in oilseeds. Exemplarypromoters include a glutelin (Gt1) promoter, which effects geneexpression in the outer layer of the endosperm, and a globulin (Glb)promoter, which effects gene expression in the center of the endosperm.Promoter sequences for regulating transcription of gene coding sequencesoperably linked thereto include naturally-occurring promoters, orregions thereof capable of directing seed-specific transcription, andhybrid promoters, which combine elements of more than one promoter.Methods for construction such hybrid promoters are well known in theart.

In some cases, the promoter is native to the same plant species as theplant cells into which the chimeric nucleic acid construct is to beintroduced. In other embodiments, the promoter is heterologous to theplant host cell.

Alternatively, a seed-specific promoter from one type of monocot may beused regulate transcription of a nucleic acid coding sequence from adifferent monocot or a non-cereal monocot.

In addition to encoding the protein of interest, the expression cassetteor heterologous nucleic acid construct includes DNA encoding a signalpeptide that allows processing and translocation of the protein, asappropriate. Exemplary signal sequences are those sequences associatedwith the monocot maturation-specific genes: glutelins, prolamines,hordeins, gliadins, glutenins, zeins, albumin, globulin, AOP glucosepyrophosphorylase, starch synthase, branching enzyme, Em, and lea.Exemplary sequences encoding a signal peptide for a protein storage bodyare identified herein as SEQ ID NOS:13-19.

In one embodiment, the method is directed toward the localization ofproteins in an endosperm cell, in some embodiments an endosperm-cellorganelle, such as a protein storage body, mitochondrion, endoplasmicreticulum, vacuole, chloroplast or other plastidic compartment. Forexample, when proteins are targeted to plastids, such as chloroplasts,in order for expression to take place the construct also employs the useof sequences to direct the gene product to the plastid, Such sequencesare referred to herein as chloroplast transit peptides (CTP) or plastidtransit peptides (PTP). In this manner, when the gene of interest is notdirectly inserted into the plastid, the expression constructadditionally contains a gene encoding a transit peptide to direct thegene of interest to the plastid. The chloroplast transit peptides may bederived from the gene of interest, or may be derived from a heterologoussequence having a CTP. Such transit peptides are known in the art. (See,for example, Von Heijne et al., 1991 Plant Mol. Biol. Rep., 9:104-126;and U.S. Pat. Nos. 4,940,835 and 5,728,925). Additional transit peptidesfor the translocation of the protein to the endoplasmic reticulum (ER)(Chrispeels K., Ann. Rev. Plant Phys. Plant Mol. Biol., 42:21-53, 1991),nuclear localization signals (Shieh et al., Plant Physiol. 1993February; 101(2): 353-361; Varagona et al., Plant Cell 1992 October;4(10): 1213-1227) or vacuole (Raikhel N., Plant Phys., 100:1627-1632,1992; and U.S. Pat. No. 5,360,726) may also find use in the constructsof the present disclosure.

Another exemplary class of signal/targeting/transport sequences aresequences effective to promote secretion of heterologous protein fromaleurone cells during seed germination, including the signal sequencesassociated with alpha-amylase, protease, carboxypeptidase, endoprotease,ribonuclease, DNase/RNase, (1-3)-beta-glucanase,(1-3)(1-4)-beta-glucanase, esterase, acid phosphatase, pentosamine,endoxylanase, β-xylopyranosidase, arabinofuranosidase, beta-glucosidase,(1-6)-beta-glucanase, perioxidase, and lysophospholipase.

Since many protein storage proteins are under the control of amaturation-specific promoter, and this promoter is operably linked to asignal sequence for targeting to a protein body, the promoter and signalsequence can be isolated from a single protein-storage gene, thenoperably linked to a transferrin protein in the chimeric geneconstruction. One exemplary promoter-signal sequence combination isexemplified in the sequence identified by SEQ ID NO:4, in which thepromoter and signal sequence both come from the rice Gt1 gene regulatoryregion. Alternatively, the promoter and leader sequence may be derivedfrom different genes. One exemplary promoter-signal sequence combinationis the rice Glb promoter linked to the rice Gt1 leader sequence (SEQ IDNO:5).

Expression vectors or heterologous nucleic acid constructs designed foroperation in plants comprise companion sequences upstream and downstreamto the expression cassette. The companion sequences are of plasmid orviral origin and provide necessary characteristics to the vector topermit the vector to move DNA from a secondary host to the plant host,such as, sequences containing an origin of replication and a selectablemarker. Typical secondary hosts include bacteria and yeast.

In one embodiment, the secondary host is E. coli, the origin ofreplication is a CoIE1-type, and the selectable marker is a geneencoding ampicillin resistance. Such sequences are well known in the artand are commercially available as well (e.g., Clontech, Palo Alto,Calif.; Stratagene, La Jolla, Calif.

The transcription termination region may be taken from a gene where itis normally associated with the transcriptional initiation region or maybe taken from a different gene. Exemplary transcriptional terminationregions include the NOS terminator from Agrobacterium Ti plasmid and therice α-amylase terminator.

Polyadenylation tails may also be added to the expression cassette tooptimize high levels of transcription and proper transcriptiontermination, respectively. Polyadenylation sequences include, but arenot limited to, the Agrobacterium octopine synthetase signal, or thenopaline synthase of the same species.

Suitable selectable markers for selection in plant cells include, butare not limited to, antibiotic resistance genes, such as, kanamycin(nptll), G418, bleomycin, hygromycin, chloramphenicol, ampicillin,tetracycline, and the like. Additional selectable markers include a bargene which codes for bialaphos resistance; a mutant EPSP synthase genewhich encodes glyphosate resistance; a nitrilase gene which confersresistance to bromoxynil; a mutant acetolactate synthase gene (ALS)which confers imidazolinone or sulphonylurea resistance; and amethotrexate resistant DHFR gene.

The particular marker gene employed is one which allows for selection oftransformed cells as compared to cells lacking the DNA which has beenintroduced. The selectable marker gene is one which facilitatesselection at the tissue culture stage, e.g., a kanamyacin, hygromycin orampicillin resistance gene.

The vectors of the present disclosure may also be modified to includeintermediate plant transformation plasmids that contain a region ofhomology to an Agrobacterium tumefaciens vector, a T-DNA border regionfrom Agrobacterium tumefaciens, and chimeric genes or expressioncassettes (described above). Further, the vectors may comprise adisarmed plant tumor inducing plasmid of Agrobacterium tumefaciens.

In general, a selected nucleic acid sequence is inserted into anappropriate restriction endonuclease site or sites in the vector.Standard methods for cutting, ligating and transformation into asecondary host cell, known to those of skill in the art, are used inconstructing vectors for use in the present disclosure. (See generally,Maniatis et al. Molecular Cloning: A Laboratory Manual, 2nd Edition,1989; Ausubel et al. Current Protocols in Molecular Biology, John Wiley& Sons, New York, N.Y., 1993; and Gelvin et al., eds. Plant MolecularBiology Manual, 1990).

Plant cells or tissues are transformed with expression constructs(heterologous nucleic acid constructs, e.g., plasmid DNA into which thegene of interest has been inserted) using a variety of standardtechniques. Effective introduction of vectors in order to facilitateenhanced plant gene expression is an important aspect of the disclosure.The vector sequences may be stably transformed, and may be integratedinto the host genome.

The method used for transformation of host plant cells is not criticalto the present disclosure. The skilled artisan will recognize that awide variety of transformation techniques exist in the art, and newtechniques are continually becoming available. Any technique that issuitable for the target host plant may be employed within the scope ofthe present disclosure. For example, the constructs can be introduced ina variety of forms including, but not limited to, as a strand of DNA, ina plasmid, or in an artificial chromosome. The introduction of theconstructs into the target plant cells can be accomplished by a varietyof techniques, including, but not limited to calcium-phosphate-DNAco-precipitation, electroporation, microinjection,Agrobacterium-mediated transformation, liposome-mediated transformation,protoplast fusion or microprojectile bombardment (Christou, 1992;Sanford et al., 1993). The skilled artisan can refer to the literaturefor details and select suitable techniques for use in the presentlydisclosed.

When Agrobacterium is used for plant cell transformation, a vector isintroduced into the Agrobacterium host for homologous recombination withT-DNA or the Ti- or Ri-plasmid present in the Agrobacterium host The Ti-or Ri-plasmid containing the T-DNA for recombination may be armed(capable of causing gall formation) or disarmed (incapable of causinggall formation), the latter being permissible, so long as the vir genesare present in the transformed Agrobacterium host The armed plasmid cangive a mixture of normal plant cells and gall.

In some instances where Agrobacterium is used as the vehicle fortransforming host plant cells, the expression or transcription constructbordered by the T-DNA border region(s) is inserted into a broad hostrange vector capable of replication in E. coli and Agrobacterium,examples of which are described in the literature, for example pRK2 orderivatives thereof. See, for example, Ditta et al., 1980 and EPA 0 120515. Alternatively, one may insert the sequences to be expressed inplant cells into a vector containing separate replication sequences, oneof which stabilizes the vector in E. coli, and the other inAgrobacterium. See, for example, McBride and Summerfeit 1990, whereinthe pRiHRI (Jouanin, et al., 1985), origin of replication is utilizedand provides for added stability of the plant expression vectors in hostAgrobacterium cells.

Included with the expression construct and the T-DNA is one or moreselectable marker coding sequences which allow for selection oftransformed Agrobacterium and transformed plant cells. A number ofantibiotic resistance markers have been developed for use with plantcells, these include genes inactivating antibiotics such as kanamycin,the aminoglycoside G418, hygromycin, or the like. The particular markeremployed is not essential to this disclosure, with a particular markerpreferred depending on the particular host and the manner ofconstruction.

For Agrobacterium-mediated transformation of plant cells, explants areincubated with Agrobacterium for a time sufficient to result ininfection, the bacteria killed, and the plant cells cultured in anappropriate selection medium. Once callus forms, shoot formation can beencouraged by employing the appropriate plant hormones in accordancewith known methods and the shoots transferred to rooting medium forregeneration of plants. The plants may then be grown to seed and theseed used to establish repetitive generations and for isolation of therecombinant protein produced by the plants.

There are a number of possible ways to obtain plant cells containingmore than one expression construct. In one approach, plant cells areco-transformed with a first and second construct by inclusion of bothexpression constructs in a single transformation vector or by usingseparate vectors, one of which expresses desired genes. The secondconstruct can be introduced into a plant that has already beentransformed with the first expression construct, or alternatively,transformed plants, one having the first construct and one having thesecond construct, can be crossed to bring the constructs together in thesame plant.

In one embodiment, the plants used in the methods of the presentdisclosure are derived from members of the taxonomic family known as theGramineae. This includes all members of the grass family of which theedible varieties are known as cereals. The cereals include a widevariety of species such as wheat (Triticum sps.), rice (Oryza sps.)barley (Hordeum sps.) oats, (Avena sps.) rye (Secale sps.), corn (maize)(Zea sps.) and millet (Pennisettum sps.). In practicing the presentdisclosure, exemplary grains are rice, wheat, maize, barley, rye andtriticale.

In order to produce transgenic plants that express transferrin proteinin seeds, monocot plant cells or tissues derived from them aretransformed with an expression vector comprising the coding sequence fora transferrin protein. The transgenic plant cells are cultured in mediumcontaining the appropriate selection agent to identify and select forplant cells which express the heterologous nucleic acid sequence. Afterplant cells that express the heterologous nucleic acid sequence areselected, whole plants are regenerated from the selected transgenicplant cells. Techniques for regenerating whole plants from transformedplant cells are generally known in the art Transgenic plant lines, e.g.,rice, wheat, corn or barely, can be developed and genetic crossescarried out using conventional plant breeding techniques.

Transformed plant cells are screened for the ability to be cultured inselective media having a threshold concentration of a selective agent.Plant cells that grow on or in the selective media are typicallytransferred to a fresh supply of the same media and cultured again. Theexplants are then cultured under regeneration conditions to produceregenerated plant shoots. After shoots form, the shoots are transferredto a selective rooting medium to provide a complete plantlet. Theplantlet may then be grown to provide seed, cuttings, or the like forpropagating the transformed plants. The method provides for efficienttransformation of plant cells and regeneration of transgenic plants,which can produce a recombinant transferrin protein.

The expression of the recombinant transferrin protein may be confirmedusing standard analytical techniques such as Western blot, ELISA, PCR,HPLC, NMR, or mass spectroscopy, together with assays for a biologicalactivity specific to the particular protein being expressed.

A purified transferrin protein recombinantly produced in a plant cell,in some embodiments mostly free of contaminants of the host plant cellis also provided. In some embodiments, the presence or absence of plantglycosyl groups can indicate that the transferrin protein was producedin a plant, but does not significantly impair the biological activity ofthe transferrin protein in any of the applied therapeutic contexts (suchthat, for example, the recombinant TG has less than a 25% loss ofactivity or less than 10% loss of activity, as compared to acorresponding non-recombinant transferrin protein). Typically, inaccordance with some embodiments, the transferrin protein constitutes atleast about 0.1%, at least about 0.5%, at least about 1.0% or at leastabout 2.0% of the total soluble protein (TSP) in the seeds harvestedfrom the transgenic plant. In some embodiments, however, proteinexpression is much higher than previously reported, i.e., at least about3.0%, which makes commercial production quite feasible. Advantageously,protein expression is at least about 5.0%, at least about 10%, at leastabout 15%, at least about 20%, at least about 30%, or even at leastabout 40% of total soluble protein.

A plant seed product prepared from the harvested seeds is also providedin the present disclosure. Preferably, the transferrin proteinconstitutes at least about 3.0% of the total soluble protein in the seedproduct, more preferably at least about 5.0%, and most preferably atleast about 10.0%. As shown in the figures, the expression oftransferrin proteins in rice grains, represented by AAT, the threefibrinogen polypeptides and HSA represent at least about 10% of totalsoluble protein.

The present disclosure also provides compositions comprising transferrinproteins produced recombinantly in the seeds of monocot plants, andmethods of making such compositions.

In practicing the disclosed method, a transferrin protein is produced inthe seeds or grain of transgenic plants that express the nucleic acidcoding sequence for the transferrin protein. After expression, thetransferrin protein may be provided to a patient in substantiallyunpurified form (i.e., at least 10-20% of the composition comprisesplant material), or the transferrin protein may be isolated or purifiedfrom a product of the mature seed (e.g., a flour, extract, malt or wholeseed composition, etc.) and formulated for delivery to a patient.

Such compositions can comprise a formulation for the type of deliveryintended. Delivery types can include, e.g. parenteral, enteric,inhalation, intranasal or topical delivery. Parenteral delivery caninclude, e.g. intravenous, intramuscular, or suppository. Entericdelivery can include, e.g. oral administration of a pill, capsule, orother formulation made with a non-nutritionalpharmaceutically-acceptable excipient, or a composition with a nutrientfrom the transgenic plant, for example, in the grain extract in whichthe protein is made, or from a source other than the transgenic plant.Such nutrients include, for example, salts, saccharides, vitamins,minerals, amino acids, peptides, and proteins other than the transferrinprotein. Intranasal and inhalant delivery systems can include spray oraerosol in the nostrils or mouth. Topical delivery can include, e.g.creams, topical sprays, or salves. Preferably, the composition issubstantially free of contaminants of the transgenic plant, preferablycontaining less than 20% plant material, more preferably less than 10%,and most preferably, less than 5%. The preferable route ofadministration is enteric, and preferably the composition isnon-nutritional.

The transferrin protein can be purified from the seed product by a modeincluding grinding, filtration, heat, pressure, salt extraction,evaporation, or chromatography.

The transferrin proteins produced in accordance with the disclosure alsoinclude all variants thereof, whether allelic variants or syntheticvariants. A “variant” transferrin protein-encoding nucleic acid sequencemay encode a variant transferrin protein amino acid sequence that isaltered by one or more amino acids from the native transferrin proteinsequence, preferably at least one amino acid substitution, deletion orinsertion. The nucleic acid substitution, insertion or deletion leadingto the variant may occur at any residue within the sequence, as long asthe encoded amino acid sequence maintains substantially the samebiological activity of the native transferrin protein. In anotherembodiment, the variant transferrin protein nucleic acid sequence mayencode the same polypeptide as the native sequence but, due to thedegeneracy of the genetic code, the variant has a nucleic acid sequencealtered by one or more bases from the native polynucleotide sequence.

The variant nucleic acid sequence may encode a variant amino acidsequence that contains a “conservative” substitution, wherein thesubstituted amino acid has structural or chemical properties similar tothe amino acid which it replaces and physicochemical amino acid sidechain properties and high substitution frequencies in homologousproteins found in nature (as determined, e.g., by a standard Dayhofffrequency exchange matrix or BLOSUM matrix). In addition, oralternatively, the variant nucleic acid sequence may encode a variantamino acid sequence containing a “non-conservative” substitution,wherein the substituted amino acid has dissimilar structural or chemicalproperties to the amino acid it replaces. Standard substitution classesinclude six classes of amino acids based on common side chain propertiesand highest frequency of substitution in homologous proteins in nature,as is generally known to those of skill in the art and may be employedto develop variant transferrin protein-encoding nucleic acid sequences.

A transferrin protein-encoding nucleotide sequence may be engineered inorder to alter the transferrin protein coding sequence for a variety ofreasons, including but not limited to, alterations which modify thecloning, processing and/or expression of the transferrin protein by acell.

As will be understood by those of skill in the art, in some cases it maybe advantageous to use a transferrin protein-encoding nucleotidesequences possessing non-naturally occurring codons. Codons preferred bya particular eukaryotic host can be selected, for example, to increasethe rate of transferrin protein expression or to produce recombinant RNAtranscripts having desirable properties, such as a longer half-life,than transcripts produced from naturally occurring sequence. As anexample, it has been shown that codons for genes expressed in rice arerich in guanine (G) or cytosine (C) in the third codon position (Huanget al., 1990). Changing low G+C content to a high G+C content has beenfound to increase the expression levels of foreign protein genes inbarley grains (Horvath et al., 2000). The transferrin protein encodinggenes can be synthesized by Operon Technologies (Alameda, Calif. basedon the rice gene codon bias (Huang et al., 1990) along with theappropriate restriction sites for gene cloning. These ‘codon-optimized’genes are then linked to regulatory/secretion sequences forseed-directed monocot expression and these chimeric genes then insertedinto the appropriate plant transformation vectors.

Heterologous nucleic acid constructs may include the coding sequence fora transferrin protein (i) in isolation; (ii) in combination withadditional coding sequences; such as fusion protein or signal peptide,in which the transferrin protein coding sequence is the dominant codingsequence; (iii) in combination with non-coding sequences, such asintrons and control elements, such as promoter and terminator elementsor 5′ and/or 3′ untranslated regions, effective for expression of thecoding sequence in a suitable host; and/or (iv) in a vector or hostenvironment in which the transferrin protein coding sequence is aheterologous gene.

Depending upon the intended use, an expression construct may contain thenucleic acid sequence encoding the entire transferrin protein, or aportion thereof. For example, where transferrin protein sequences areused in constructs for use as a probe, it may be advantageous to prepareconstructs containing only a particular portion of the transferrinprotein encoding sequence, for example a sequence which is discovered toencode a highly conserved transferrin protein region.

In some embodiments, a seed composition containing a flour, extract, ormalt obtained from mature monocot seeds and one or more seed-producedtransferrin proteins in unpurified form is provided. Isolating thetransferrin proteins from the flour can entail forming an extractcomposition by milling seeds to form a flour, extracting the flour withan aqueous buffered solution, and optionally, further treating theextract to partially concentrate the extract and/or remove unwantedcomponents. In a preferred method, mature monocot seeds, such as riceseeds, are milled to a flour, and the flour then suspended in saline orin a buffer, such as Phosphate Buffered Saline (“PBS”), ammoniumbicarbonate buffer, ammonium acetate buffer or Tris buffer. A volatilebuffer or salt, such as ammonium bicarbonate or ammonium acetate mayobviate the need for a salt-removing step, and thus simplify the extractprocessing method.

In some embodiments, the level of protein expressed in a transgenicplant is assessed from a crude extract or substantially unpurifiedcomposition from the plant seed. In some embodiments, a grain or milledgrain or flour composition, an extract composition, or malt compositionobtained from mature monocot seeds is produced in substantiallyunpurified form. The transferrin protein may be present in an amountbetween about 0.05 and 0.5 grams protein/kg total soluble protein. For agrain composition, the level of transferrin protein present may bebetween 0.1 to 1% of total seed weight. For an extract composition, thetransferrin protein may be concentrated to form up to 5-40% or more ofthe total extract weight. A malt composition, which will contain asignificant percent of malt sugars, in addition to native proteins aswell as heterologous transferrin protein, will typically contain anamount of protein that is intermediate between that of grain and theextract.

The flour suspension is incubated with shaking for a period typicallybetween 30 minutes and 4 hours, at a temperature between 20-55° C., Theresulting homogenate is clarified either by filtration orcentrifugation. The clarified filtrate or supernatant may be furtherprocessed, for example by ultrafiltration or dialysis or both to removecontaminants such as lipids, sugars and salt. Finally, the materialmaybe dried, e.g., by lyophilization, to form a dry cake or powder. Theextract combines advantages of high protein yields, essentially limitinglosses associated with protein purification,

In general, the protein once produced in a product of a mature seed canbe further purified by standard methods known in the art, such as byfiltration, affinity column, gel electrophoresis, and other suchstandard procedures. The purified protein can then be formulated asdesired for delivery to a human patient. More than one protein can becombined for the therapeutic formulation. The protein may be purifiedand used in biomedical applications requiring a non-food administrationof the protein.

Illustrative publications describing components of precursorcompositions, as well as methods for preparing certain compositionsinclude the following: U.S. patent Ser. Nos. 12/751,869 and 12/558,189;U.S. Patent Application Publication Nos. 20080318277; 20090156486;20090258004; 20100031394 and 20030056244, and U.S. Pat. Nos. 6,991,824;7,417,178 and 7,589,252 each of which is incorporated by referenceherein in its entirety.

EXAMPLES

This section will describe the various different working examples thatwill be used to highlight features of the present disclosure. However,the present disclosure shall in no way be considered to be limited tothe particular embodiments described below.

Example 1 Development of hTF Expression Vector and Plant Transformation

To obtain high level expression of rhTF in rice seeds, the mature hTFprotein amino acid sequence (Swiss-Prot accession number P02787, setforth as SEQ ID NO: 3) was back translated into a nucleotide sequencewith the codons optimized towards the codon-usage preference of ricegenes (http://www.kazusa.or.jp/codon). At the same time, internalrepeats and other features that might affect mRNA stability ortranslation efficiency were avoided. Compared to the native genesequence for mature hTF, nucleotides in 339 out of a total of 679 codonswere modified in the codon-optimized nucleotide sequence for hTF withoutaltering the encoded amino acid sequence, and the G+C content wasincreased to 65% from 50.6% in the native hTF gene sequence. Tofacilitate the subcloning of hTF gene into an expression vector, theMlyI blunt-cutting restriction site that allows a cut right before thefirst nucleotide of the hTF gene was engineered, while two consecutivestop codons followed by an XhoI restriction site were engineered afterthe last genetic codon of hTF gene. The entire gene sequence wassynthesized by the company DNA2.0 (Menlo Park, Calif.).

The synthesized nucleotide sequence for rhTF was digested with MlyI andXhoI, and ligated in frame into the NaeI-XhoI sites of the expressionvector pAPI 405; and thereby the hTF gene is operably linked to thedownstream of rice seed storage protein glutelin 1 gene promoter (GU)including its signal peptide encoding sequence (GenBank accession no.Y00687) and to the upstream of the nopaline synthase (nos) geneterminator of Agrobacterium tumefaciens. The resulting plasmid wasvalidated by sequencing in both orientations, and designated as pVB24.

The plasmid pAPI146 was used to provide a selection marker in planttransformation. The pAPI146 consists of the hpt (hygromycin Bphosphor-transferase) gene encoding the hygromycin B-resistant proteinunder the control of rice beta-glucanase 9 gene promoter, whichrestricts the expression of hpt gene only in rice calli (Huang, et al.,Plant Science 161: 589-95 (2001)). The linear expression cassette DNAfragments comprising the region from promoter to terminator (without thesuperfluous backbone plasmid sequence) (See FIG. 1) in both pVB24 andpAPI146 plasmids were prepared by double digestion of EcoRI and HindIII,and used for transformation. Microprojectile bombardment-mediatedtransformation of embryonic calli induced from the mature seeds of twocultivars, Tapei309 and Bengal (Oryza sativa L. subsp. Japonica), wasperformed as described previously (Huang, et al., Plant Science 161:589-95 (2001)). Before the regenerated transgenic seedlings weretransferred to soil, PCR analysis of the plants were conducted withprimers specific to the hTF gene using the Extract-N-Amp Plant PCR kit(Sigma, St. Louis, Mo.), and plants shown as negative were discarded.The regenerated transgenic plants are referred to as R₀ plants ortransgenic events, and their progeny in successive generations aredesignated as R₁, R₂, etc.

Example 2 Expression Analysis of Recombinant hTF from Transgenic Rice

To identify transgenic events expressing rhTF, pooled R₁ seeds of eachtransgenic event (R₀) were analyzed because of the genetic segregationof hemizygous hTF gene in the selfed R₁ seeds. Eight R₁ seeds from eachtransgenic event were randomly picked, dehusked, and placed into eightwells in the same column of a 96 deep-well plate. Five hundredmicroliters of PBS buffer (pH 7.4) and two 2 mm diameter steel beadswere dispensed into each well. Then, a homogeneous extract was producedby agitating the plate with a Geno/Grinder 2000 (SPEX CertiPrep,Metuchen, N.J.) for 20 min at 1300 strokes/min followed bycentrifugation with a microplate centrifuge at 4,000 rpm for 20 min.Equal amounts of supernatant extract from each seed of the sametransgenic event were pooled. Two microliters of the pooled proteinextracts from each transgenic event were spotted onto a nitrocellulosemembrane. The blot was blocked in 5% non-fat milk in Tris bufferedsaline tween-20 (TBST) buffer for 1 h, and then incubated with rabbitanti-hTF antibody (Abcam, Cambridge, Mass.) in TBST buffer at aconcentration of 1 μg/ml for 1 h followed by washing 4 times (5 mineach) with TBST buffer. Then, the blots were incubated with 1:20,000diluted anti-rabbit HRP (horseradish peroxidase)-conjugated antibody(BioRad, Hercules, Calif.) in TBST buffer for 1 h followed by 3 washes,5 min each in TBST buffer, and one wash in TBS buffer for 5 min. The dotblots were then incubated with the enhanced chemiluminescence (ECL)reagent (Perice Biotechnology, Rockford, Ill.) for 5 min, and thenexposed to X-ray film for signal detection (See FIG. 2).

The seed protein extracts from positive transgenic plants identified byimmuno-dot blot were resolved on a 4-20% Tris-glycine SDS-PAGE gel,electro-blotted onto a 0.45 um nitrocellulose membrane for 1 h at 100Vin a Bio-Rad Protean System (BioRad, Hercules, Calif.). The subsequentwestern blot detection procedure was the same as described fordot-immunoblot except that the secondary antibody was the anti-rabbitalkaline phosphatase-conjugated antibody (BioRad, Hercules, Calif.) at a1:4000 dilution and that the blot was developed with BCIP/NBT substrate(Sigma, St. Louis, Mo.).

In total, 195 independent fertile transgenic rice plants (R₀) weregenerated from the particle bombardment transformation of two ricecultivars, Bengal and Taipei 309, by using linear rhTF gene expressioncassette DNA (FIG. 1). The expression screening analysis of R₁ seedsthrough immuno dot-blot assay of protein extracts showed that 54 plantsexhibited detectable expression of rhTF (FIG. 2). Rice seed TSP wasextracted with 0.5 ml/seed of PBS buffer, pH 7.5 at room temperature for1 h followed by centrifugation. 2 μl each of pooled protein extract fromeach transgenic event were spotted onto a nitrocellulose membrane. Spotsin rows A to F and columns 1-12 are TSP extracts from 72 transgenic riceevents. Spots G1-6 and G7-12 are TSP extracts from non-transgenic ricecultivars Bengal and Tapei309, respectively. Spots Hl-6 are 10, 20, 50,100, 200 and 500 ηg of nhTF (Sigma) spiked into 2 μl of Bengal seedprotein extract, respectively. The spots H7-12 contained 10, 20, 50,100, 200 and 500 ηg of nhTF (Sigma) spiked into 2 μl of Taipei309 seedprotein extract, respectively. FIG. 3 shows SDS-PAGE and immunoblotanalysis of rhTF expressed in rice grain. Total soluble protein TSP wasextracted (but not concentrated, enriched or purified) from rice flourof transgenic lines expressing rhTF and non-transgenic line Bengal with25 mM Tris-HCL pH 7.5 at a 1:10 ratio (g/ml) of buffer to rice flour.TSP was directly loaded and resolved on two 4-20% Tris-glycine SDS-PAGEgels (Invitrogen). One gel was stained with Coomassie blue (FIG. 3A),and the other was used for western blot immuno-detection with anti-hTFantibody (FIG. 3B). The arrowhead indicates the protein bandscorresponding to rhTF. M=Molecular weight standard; lane 1=20 μg of nhTF(Sigma); lane 2=wild-type Bengal seed protein extract; lanes3-8=transgenic events VB24-17, 54, 57, 401, 77 and 136, respectively.The SDS-PAGE analysis revealed a predominant protein band correspondingto the molecular weight of native hTF in positive transgenic seeds butnot in the wild-type rice seeds (FIG. 3A), and the band was shown tospecifically cross-react with anti-hTF antibody (FIG. 3B).

The transgenic events with high level expression of rhTF were identifiedthrough the denstometric analysis of the immuno dot signals followed byELISA quantification. The expression level of rhTF in R₁ seeds was shownto be about 40% of total soluble protein (TSP). However, the measurementof rhTF expression level as a percent of TSP varied significantlydepending on different extraction buffers and conditions used becausethe extracted amount of native rice seed proteins was significantlyimpacted by pH, ionic strength, and temperature (data not shown).Therefore, the percent of biomass dry weight represented by rhTF is amore reliable estimate of rhTF expression level. The expression level ofrhTF in some selected transgenic events was up to 8.8 mg per gram(0.088%) of dry R₁ seed; and reached over 10 mg per gram (1%) of seeddry weight at R₂ generation and remained stable in subsequent generation(Table 1). The relatively lower expression level of rhTF in R₁ seedscompared to that in subsequent generation seeds is likely because of thepoor plant growth performance and seed development of R₀ plants. Similarobservations have been reported by others (Hood, et al., MolecularBreeding 3 (1997) 291-306; Chikwamba, et al., Transgenic Research 11(2002) 479-493). Data are shown in Table 1, below.

TABLE 1 Quantification of rhTF expression levels over three generationsin rice grains VB24-17 VB24-54 VB24-57 Generation n Mean ± Std n Mean ±Std n Mean ± Std R₁ ^(a) 8  8.8 ± 0.9 8  8.0 ± 0.8 8  7.7 ± 0.3 R₂ ^(b)59 10.2 ± 1.7 64 10.0 ± 1.7 76 10.1 ± 2.1 R₃ ^(c) 10 10.5 ± 1.8 10 10.5± 1.4 15 10.1 ± 1.6 ^(a)Eight R₁ positive seeds from each transgenicevent were assayed ^(b)One gram of pooled R₂ seeds from a singleTF-positive R₁ plant was assayed ^(c)One gram of pooled R₃ seeds fromeach single homozygous R₂ plant was assayed

Quantification of rhTF was performed by ELISA (enzyme-linkedimmunosorbent assay) with a hTF ELISA assay kit (Bethyl Labs,Montgomery, Tex.) by following the manufacturer's instructions, exceptthat the purified hTF from Sigma was used to produce the standard curve.Low expression yield of recombinant proteins has been identified as oneof the major limitations of plant expression systems (Lienard, et al.,Biotechnol. Annu. Rev. 13 (2007) 115-47; Fischer, et al., Curr. Opin.Plant Biol. 7 (2004) 152-8), and Farran et al. (2002) suggested that thecritical limit of plant-derived recombinant protein expression level forcommercial viability is 0.01% mass weight (Farran, et al., TransgenicRes. 11 (2002) 337-46). The rice-derived rhTF expression level was 100fold higher than this suggested critical limit. This extremely highexpression level will contribute to significantly reduce the productioncost, and will also benefit the downstream purification.

To investigate the tissue-specificity of rhTF expression, proteins wereextracted from roots, stems, leaves, leaf sheaths, anthers with pollens,grain husks, pistils, immature seeds, and mature seeds, respectively,with PBS buffer (pH 7.4), resolved on two 4-20% Tris-glycine SDS-PAGEgels (Invitrogen), run simultaneously, and stained with LabSafe Gel Blue(G Biosciences) (FIG. 4A), or transferred to a membrane forimmunodetection using anti-hTF antibody (FIG. 4B) as described above.Lanes 1−9=10 μg per lane crude protein extract from roots, stems,leaves, leaf sheaths, anthers with pollens, grain husks, pistils,immature seeds, and mature seeds, respectively. Lane 10=4 μg ofcommercial native hTF (Sigma), indicated by arrowhead. The analysis ofthe tissue specificity of rhTF expression demonstrated that the rhTF wasexpressed only in the maturing and mature seeds, but not in the root,stem, leaf, leaf sheath, grain husk, anther including pollen, and thepistils (FIG. 4). This is consistent with previous finding that the Gt1gene promoter is developmentally regulated and active only in maturingrice seeds (Okita, et al., J. Biol. Chem. 264 (1989) 12573-81; Qu le, etal., Plant Biotechnol. J. 2 (2004) 113-25).

Example 3 Extraction and Purification of rhTF

Identification of the optimal extraction conditions for rhTF isimportant for developing a purification procedure that allows maximalprotein purity and minimal purification costs. To find the optimalextraction condition for rhTF, the effect of temperature, buffer pH,ionic strength, and mixing time on protein extraction was investigatedusing 100 mg of rice seed flour in each treatment. The temperatureeffect on rhTF extraction was examined by extracting 100 mg of rice seedflour in 1 ml of PBS buffer, pH 7.4 at room temperature (RT), 37° C.,40° C., or 60° C., for 1 h. The effect of buffer pH on rhTFextractability was tested in a range from 4.5 to 10.0. The rice seedflour was extracted in each Eppendorf tube with 1 ml of 25 mM sodiumacetate at pH 4.5, 5.0, 6.0; 25 mM Tris-HCl at pH 7.0, 7.5, 8.0, 9.0; or25 mM CAPS, pH 10.0 for 1 h at RT. The ionic strength effect on rhTFextraction was determined by extracting 100 mg of rice flour in each of1 ml 25 mM Tris-HCl, pH 8.0 with 100, 200, or 500 mM sodium chloride for1 h at RT. The time effect on rhTF extraction was determined byextracting 100 mg of rice flour in 1 ml of 25 mM Tris-HCl, pH 8.0 for10, 30, 60, or 120 min. After extraction, all samples were centrifugedat 13,000×g for 20 min, and the supernatants were assayed to estimatethe total soluble protein (TSP) and rhTF protein content.

It was shown that while the amount of TSP increased with the increase inpH, the extracted rhTF protein was shown to increase with increase in pHfrom 4.5 to 7.0 but no substantial difference in the pH range of 7.0 to10.0 (data not presented). Comparison of the effect of extraction timeshowed that 30 min extraction was already able to exact the maximumamount of rhTF. Neither the salt concentration nor the extractiontemperature showed a significant effect on the rhTF extractability (datanot shown). These results indicated that extraction of rhTF from riceflour with 25 mM Tris-HCl, pH 7.5 for 30 min at RT was the optimalcondition to maximize the extraction of rhTF while minimizing theextraction of rice native proteins.

To develop a cost-effective procedure for purification of rhTF,different chromatography media and conditions were tested. Thepurification of rhTF protein was tested with hydrophobic interactionchromatography (HIC) medium Phenyl Sepharose 6 FF, anion exchangechromatography media Q (quaternary amine) and DEAE (diethyl aminoethane) Sepharose FF (GE, Piscataway, N.J.), respectively, using theBiologic LP chromatography system (Bio-Rad, Hercules, Calif.). Each typeof chromatography media was packed to 5 cm high in a 1×10 cm Bio-RadEcono column. The purification of rhTF protein using Phenyl Sepharoseresin was carried out essentially as described in (Ali, et al., Biochem.J. 319 (Pt 1): 191-5 (1996)). For the purification of rhTF protein withanion exchange chromatography, the seed crude total proteins wereextracted with 25 mM Tris-HCl buffer, pH 7.5 at a ratio of 1 to 10 offlour to buffer (g/ml) for 30 min at RT followed by centrifugation at15,000×g for 30 min. The supernatant was filtered through a 0.2 umfilter, and then loaded onto a DEAE or Q Sepharose columnpre-equilibrated with 25 mM Tris-HCl buffer, pH 7.5. After the columnwas washed with 25 mM Tris-HCl buffer, pH 7.5 to the UV and conductivitybaseline, the rhTF protein was eluted either by linear gradient from 0to 100 mM NaCl in 25 mM Tris-HCl buffer, pH 7.5 or by a step elutionwith 40 mM NaCl in 25 mM Tris-HCl buffer, pH 7.5.

The HIC column with a Phenyl Sepharose was shown to be able to purifyrhTF at a purity of 90%. However, a step of precipitating impureproteins with ammonium sulphate before loading onto the column couldreduce the yield of rhTF and also add the purification cost. The weakanion exchange chromatography DEAE showed that the rhTF bound to theDEAE resin in the extraction buffer 25 mM Tris-HCl, pH 7.5 without theneed of buffer exchange, while some rice proteins leaked out of theresin into the flow-through fractions during loading and washing. TherhTF could then be eluted from the DEAE resin with 40 mM NaCl in 25 mMTris-HCl, pH 7.5, and was at a purity of greater than 95% based on theSDS-PAGE (FIG. 5). The purification of rhTF with the strong anionexchange chromatography Q Sepharose resin showed a very similarchromatographic profile to that of DEAE Sepharose column. However, the QSepharose resin bound rhTF protein more strongly than DEAE Sepharoseresin, and the rhTF protein needed to be eluted with higherconcentration of salts, resulting in coeluting more rice proteins. Withthe DEAE chromatography, we purified rhTF with four batches of 100 gseed flour and each batch consistently yielded the recovery rate of rhTFto 60%. These results showed that a one-column DEAE chromatographymethod can effectively purify rhTF from rice grain protein extracts. Theease of purifying rhTF with a single purification step is presumablyenabled by both the high expression level of rhTF and the relativelysimple protein composition in rice grain (Stoger, et al., Plant Mol.Biol. 42 (2000) 583-90), because either of them will lead to a higherenrichment of target protein in the starting material for purification,which can help simplify the purification process and reduce the cost.The ease and low cost of purification of recombinant proteins from ricegrains have also been shown in our prior work with recombinantlactoferrin (Nandi, et al., Transgenic Res. 14 (2005) 237-49) andlysozyme (Huang, et al., Molecular Breeding 10 (2002) 83-94; Wilken, etal. Biotechnol. Prog. 22 (2006) 745-752).

Example 4 Amino-Terminal Sequence Analysis

Amino (N)-Terminal Sequence Analysis.

Since a rice seed storage protein signal sequence targeting to theprotein body in endosperm was fused to the N-terminus of the rhTF,N-terminal sequencing of rhTF was carried out to examine whether therice signal sequence was cleaved correctly. Eleven sequencer cycles wereanalyzed, and the N-terminal sequence of rhTF was revealed as V P D K TV R W-X^(c)-A-V (SEQ ID NO: 23), which is identical to nhTF except thatthe expected cysteine amino acid residue at cycle 9 was not determined.The undetected cysteine is expected because cysteine, without specialmodification, cannot be detected by N-terminal sequencing. This resultindicates that the rice signal sequence before the mature rhTF proteinwas correctly removed at the expected position.

The purified rhTF was resolved on a 4-20% Tris-glycine SDS-PAGE gel(Invitrogen, Carlsbad, Calif.) and electroblotted onto a PVDF membrane(Bio-Rad, Hercules, Calif.) in 50 mM CAPS buffer, pH 10.0. The blot wasstained with 0.1% Ponceau S in 0.1% acetic acid for 5 min, and destainedin 0.1% acetic acid and ddH2O. The protein band corresponding to rhTFwas excised and sequenced on an ABI 494-HT Procise Edman Sequencer atthe Molecular Structure Facility at the University of California, Davis,Calif., US.

Example 5 MALDI (Matrix-Assisted Laser Desorption Ionization) Analysisof rhTF

Molecular Weight of rhTF.

The MALDI analysis was carried out to estimate the molecular weight ofrice-derived rhTF. Three sources of TFs, rice-derived rhTF,yeast-derived aglycosylated rhTF (Millipore, Billerica, Mass.), andnative hTF (Sigma, St. Louis, Mo.), were all dialyzed against 50 mMsodium acetate, 5 mM EDTA, pH 4.9 overnight followed by dialyses inddH₂O to deplete iron that was bound to TFs. These iron-free or apo-TFswere further desalted using ZipTip™μ-C18 pipette tips (Millipore,Billerica, Mass.), eluted with a solution of 70% acetonitrile (ACN),0.2% formic acid, and 5 mg/ml MALDI matrix (α-cyano-4-hydroxycinnamicacid), and spotted onto the MALDI target and analyzed with an AppliedBiosystems 4700 Proteomics Analyzer (Applied Biosystems Inc., FosterCity, Calif.) at the Molecular Structure Facility at the University ofCalifornia, Davis, Calif., US.

A close-up view of the MALDI spectrum of rhTF revealed a peak comprisingtwo small split peaks on top with molecular weights of 75,255.6 and76,573.8 Da, respectively (FIG. 6). This MALDI spectrum is similar tothat of the yeast-derived aglycosylated rhTF but different from theN-glycosylated nhTF spectrum, which showed a single peak of 80,000 Damass (Data not shown). The mass for the first split small peak of therice-derived rhTF is close to the calculated mass of non-N-glycosylatednhTF (75,181.4 Da) with a mass shift of just 74.2 Da, and the mass forthe second split small peak showed a mass increase of 1,392.4 Da. Thesize discrepancy between rhTF and N-glycosyalted nhTF as revealed byMALDI is consistent with the finding as shown in the SDS-PAGE gelanalysis of rhTF (FIG. 5). Furthermore, the rice-derived rhTF molecularweight as revealed by MALDI is similar with that of the yeast-derivedaglycosylated rhTF, suggesting that the rice-derived rhTF may not beN-glycosylated.

Example 6 PNGase F Digestion of rhTF

Glycosylation Modifications.

To evaluate the glycosylation status of rice-derived rhTF, the purifiedrhTF protein was subjected to digestion with peptide-N-glycosidase F(PNGase F) (Sigma, St. Louis, Mo.). The yeast-derived aglycosylated rhTF(Millipore, Billerica, Mass.) and native hTF (Sigma, St. Louis, Mo.)were also included for comparison (FIG. 7). The native hTF contains twoN-glycosylation sites (N413 and N611) (MacGillivray, et al., J. Biol.Chem. 258 (1983) 3543-53), whereas the yeast-derived aglycosylated rhTFhas two mutations of its N-glycosylation sites (N413Q and N611Q),rendering a protein without N-glycosylation (Sargent, et al., BioMetals(2006) 19:513-519).

All TFs were desalted and buffer exchanged into 20 mM ammoniumbicarbonate, pH 8.6 using 10 KDa MWCO Microcon spin columns (Millipore,Billerica, Mass.) with a final TF concentration of 0.5 mg/ml. Then, 45μl of each type of TF was aliquoted into an Eppendorf tube followed byadding 5 μl of 10× denaturant (0.2% SDS, 10 mM 2-mercaptoethanol, 20 mMammonium bicarbonate, pH 8.6) and boiling for 10 min. After the sampleswere cooled to RT, 5 μl of 15% Triton X-100 was added followed by theaddition of 5 μl (2.5 units) PNGase F to remove the glycans from TFs.The reaction was carried out at 37° C. overnight (16 h) and analyzed byresolving 15 μl of each reaction on 4-20% Tris-glycine SDS-PAGE gel(Invitrogen, Carlsbad, Calif.) and staining with LabSafe Gel Blue (GBiosciences, St. Louis, Mo.).

As expected, the N-glycosylated nhTF showed a clear downward shift inelectrophoretic mobility after PNGase F treatment, and the yeast-derivedaglycosylated rhTF showed no change before and after the PNGase Ftreatment. Surprisingly, the electrophoretic mobility of rice-derivedrhTF also remained unchanged before and after the PNGase F treatment,and its molecular size was the same as that of deglycoslated native hTFby PNGase F and yeast-derived aglycosylated rhTF. This result isconsistent with the data revealed by MALDI analysis, and they allsuggest that rice-derived rhTF is not N-glycosylated. The absence ofN-glycosylation in rice-derived rhTF is, however, inconsistent with ourprior finding in recombinant human lactoferrin (a close relative tohTF), which is expressed in rice grain using the same expression vectorfor rhTF and shown to be N-glycosylated (Nandi, et al., Transgenic Res.14 (2005) 237-49; Nandi, et al., Plant Science 163 (2002) 713-22). Themechanism of the formation of non-N-glycosylated rhTF warrants furtherinvestigation.

Example 7 Analysis of the Isoelectric Point of rhTF

The isoelectric point of rice-derived apo-rhTF was determined with apre-cast Novex IEF (isoelectric focusing) gel, pH 3-10 (Invitrogen,Carlsbad, Calif.) according to manufacturer's instruction. Fourmicrograms of TF in dH₂O were resolved at 100 V for 1 h, 200V for 1 h,and 300 V for 30 min. The native apo-hTF (Sigma, St. Louis, Mo.) and theyeast-derived aglycosylated apo-rhTF (Millipore, Billerica, Mass.) werealso loaded on the gel for comparison. Lane 1=native hTF (Sigma); lane2=yeast-derived aglycosylated rhTF (Millipore); lane 3=rice-derivedrhTF. The gel was then fixed in 136 mM sulphosalicylic acid and 11.5%trichloroacetic acid (TCA) for 30 min and then stained in 0.1% CoomassieBrilliant Blue R-250 followed by destaining.

The isoelectric point (pI) of rice-derived rhTF was shown to be 6.3,which is same as the pI of yeast-derived aglycosylated rhTF but one unithigher than the pI of the native hTF (5.3) (FIG. 8). The pI discrepancyof rhTF and native hTF is due to the negatively charged sialic acidresidues present in the native hTF but absent in both rice-derived andyeast-derived rhTFs. The native hTF has two N-linked oligosaccharidechains, and each chain terminates in two or three antennae, each withterminal sialic acid residues (MacGillivray, et al., J. Biol. Chem. 258(1983) 3543-53; Fu, et al., Anal. Biochem. 206 (1992) 53-63). It hasbeen reported that loss of the sialic acid residues leads to a cathodicshift of the pI of TF molecules (Hoefkens, et al., Glycoconj. J. 14(1997) 289-95). The yeast-derived aglycosylated rhTF has no N-linkedglycans and sialic acid residues. The rhTF expressed in rice grain isnot expected to have sialic acids either, as plants are presumably notcapable of synthesizing sialic acids or at best just contain negligibleamounts (Castilho, et al., Plant Physiol. 147 (2008) 331-9; Zeleny, etal., Planta 224 (2006) 222-7).

Example 8 RP-HPLC Analysis of rhTF

Conformation of rhTF The conformation and integrity of rice-derivedapo-rhTF was assessed by comparing with the apo-nhTF using reverse phaseliquid chromatography (RP-HPLC).

Both native apo-hTF (Sigma, St. Louis, Mo.) and rice-derived apo-rhTFwere prepared in buffer A containing 0.1% trifluoroacetic acid (TFA) and5% ACN at a concentration of 50 μg/ml and filtered through a 0.2 umsyringe filter (PALL, Port Washington, N.Y.). Then 2.5 μg of eachprotein sample was injected to a pre-equilibrated Zorbax 3000SB-C8column (Aglient, Santa Clara, Calif.) with buffer A using a BeckmanCoulter System Gold 126 solvent module (Beckman, Fullerton, Calif.). Thecolumn was washed with three column volume of buffer A, and then runwith a gradient from buffer A to 100% buffer B containing 0.04% TFA and95% ACN in 12 column volume.

RP-HPLC resolved both the rhTF and nhTF into a major peak correspondingto their respective monomer form of the molecule, and the two peaks wereshown to have the same retention time (FIG. 9), suggesting thatrice-derived rhTF has similar conformational structure as nhTF.

Example 9 Iron-Binding Assay of rhTF

To test the reversible iron binding capacity of rice-derived rhTF, thepurified rhTF was first dialyzed against 50 mM sodium acetate, 5 mMEDTA, pH 4.9 overnight followed by sequential dialyses in ddH2O and 25mM Tris-HCl, pH 7.5 to remove the iron that was bound to rhTF. Then, theapo-rhTF at a concentration of 5 mg/ml in 25 mM Tris-HCl buffer, pH7.4+10 mM NaHCO3 was titrated with increasing amount of iron(III)-nitrilotriacetate (Fe3+-NTA). The spectra were scanned from 700 to380 nm after each addition of Fe3+-NTA, and the reading was correctedfor dilution. The iron-saturated rhTF was dialyzed in 25 mM Tris-HClbuffer, pH7.5 overnight with three buffer changes to remove the unboundiron, resulting in the holo-rhTF. The iron-binding status of rhTF withdifferent iron saturation levels was assayed by examining the mobilityof rhTF on the Urea-PAGE gel with the method as described in (Evans, etal., Biochem. J. 189: 541-46 (1980); Makey, et al., Biochim. Biophys.Acta 453 250-6 (1976)). Approximately 2 μg of each TF sample was mixedwith equal volume of 2× sample buffer (89 mM Tris-borate, pH 8.4, 7 Murea, 50% sucrose, 0.01% bromophenol blue), loaded onto a Novex precast6% TBE-Urea PAGE gel (7M urea), and electrophoresed in a buffercontaining 89 mM Tris-borate, 20 mM EDTA, pH 8.4 for 2 h at 170 V. Thegel was stained with Coomassie blue.

Results of Iron Binding Assay

The biological function of TF was measured by assessing its ability tobind and release iron reversibly. The purified, partially iron saturated(pis) rhTF from rice grains showed a salmon-pink color, a characteristiccolor of iron-bound TF, suggesting that rhTF has already bound iron inrice grains. After being dialyzed against 50 mM sodium acetate, 5 mMEDTA, pH 4.9 overnight followed by sequential dialysis in ddH₂O and 25mM Tris-HCl, pH 7.5, the pinkish rhTF became colorless (FIG. 10A), anindication of iron release from the pis-rhTF, resulting in theconversion into apo-rhTF. Apo-rhTF was titrated with increasing amountsof iron (III)-nitrilotriacetate (Fe³⁺-NTA), and the visible spectra werescanned from 700 to 380 nm after each addition of Fe³⁺-NTA and thereading corrected for dilution. Spectrophotometric titration of thisapo-rhTF with iron (Fe³⁺-NTA) showed a broad peak in the region of 465to 470 nm, and the peak grew in size as the rhTF was gradually saturatedwith the increasing increments of iron (FIG. 10D). At the same time, thepink color also gradually showed up in the titrated rhTF solution andbecame darker when rhTF was saturated with iron (FIG. 10A). Thesaturation of apo-rhTF with iron resulted in the production ofholo-rhTF.

To evaluate the iron binding status of purified pis-rhTF and its derivedapo- and holo-isoforms after iron depletion and saturation, these rhTFsamples were subjected to a urea-PAGE gel electrophoresis analysis. Theapo- and holo-rhTF both showed a single band but with slower and fasterelectrophoretic mobility, respectively, in the urea-PAGE gel (FIG. 10B).The slower and faster migrating forms of rhTF reflected theconformational change of rhTF without or with bound iron (Sargent, etal., BioMetals (2006) 19:513-519); Evans, et al., Biochem. J. 189 (1980)541-46). The pis-rhTF showed three bands in the urea-PAGE gel; theslowest and the fastest bands corresponded to the apo- and holo-forms ofrhTF, respectively, whereas the middle band represented the monoferricform of rhTF. The coexistence of apo-, holo- and monoferric-rhTF in thepurified rhTF indicated that rhTF had been indeed partially saturatedwith iron in the rice grain. The monoferric form of rhTF was furtherinferred to have an iron bound in C-lobe of rhTF because the band wasshown to be closer to the apo-rhTF, which is a characteristic ofC-terminal monoferric TF (Evans, et al., Biochem. J. 189 (1980) 541-46;Mason, et al., Protein Expr. Purif. 36 (2004) 318-26). In normal serumwith an iron concentration insufficient to saturate TF, the twomonoferric forms of hTF (C- and N-terminal) can be revealed in theurea-PAGE gel because both N- and C-terminal iron-binding sites areoccupied with iron although the N-terminal site is normallypreferentially occupied (Zak, et al., Blood 68 (1986) 157-61; Williams,et al., Biochem. J. 185 (1980) 483-488). However, when the serum isdialyzed against a buffer at pH 7.4, iron is found to preferentiallybind to the C-terminal site so that the N-terminal monoferric TF isundetectable in the urea-PAGE gel (Williams, et al., Biochem. J. 185(1980) 483-488). Similarly, the rice-derived rhTF was extracted andpurified at pH 7.5 followed by a step of dialysis at pH 7.5 toconcentrate, and thus these conditions could cause the C-terminaliron-binding site of rhTF to be predominantly occupied with iron,resulting in the absence of the band corresponding to N-terminalmonoferric rhTF.

The electrophoretic mobility of rice-derived apo- and holo-rhTF inurea-PAGE gel was compared to that of native hTF and the yeast-derivedaglycosylated rhTF (FIG. 10C; lane 1=native apo-hTF; lane2=yeast-derived aglycosylated apo-rhTF; lane 3=rice-derived apo-rhTF;lane 4=native holo-hTF; lane 5=yeast-derived aglycosylated holo-rhTF;lane 6=rice-derived holorhTF). It was shown that the rice-derived apo-or holo-rhTF migrated with the same mobility exhibited by theircorresponding form of yeast-derived aglycosylated rhTF. These resultsshowed that rice-derived rhTF was able to bind and release ironreversibly. However, both apo- and holo-native hTF exhibited fastermobility compared to their respective counterpart of recombinant hTF.The faster electrophoretic mobility of native hTF is associated with itspossession of negatively charged sialic acid residues that are absent inboth rice- and yeast-derived rhTFs.

Example 10 Cell Growth and Antibody Productivity Assay of rhTF

The rice-derived rhTF was compared to the native holo-hTF (Sigma, St.Louis, Mo.) to test its effect on proliferation and productivity ofhybridoma cells under serum-free conditions. The log phase Sp2/0-derivedhybridoma cells AE1 (ATCC HB-72) were prepared by growing in DMEM/F12medium+1% FBS+ITSE supplement (insulin 10 μg/ml, TF 5.5 μg/ml, Sodiumselenite 0.0067 μg/ml, ethanolamine 2.0 μg/ml (Invitrogen, Carlsbad,Calif.). The cells were then washed three times with DMEM/F12 withoutsupplements to remove FBS and TF, and seeded in serum-free assay medium(DMEM/F12 supplemented with ISE (no TF) and 1 g/L CELLASTIM™(recombinant human albumin) (InVitria, Fort Collins, Colo.)) at 0.8×105viable cells/ml. A dose response study was carried out by adding rhTF orits native counterpart hTF (Sigma, St. Louis, Mo.) into assay medium atconcentrations of 0.03, 0.1, 0.3, 1.0, 5.0, and 30 μg/ml and examiningtheir cell proliferation effect after three days of growth in ahumidified incubator, 37° C., 6% CO2. The negative control was the sameassay medium without any added TF, while 10% FBS and ITSE cocktail(Invitrogen, Carlsbad, Calif.) in assay medium were positive controls.The assay was carried out in duplicate 1 ml stationary cultures for eachcondition. The concentration of viable cells was determined by a GuavaPCA cell counter. The cell proliferation effect of rhTF was furtherevaluated by using cell growth curve. The AE1 cells were grown in assaymedium with the addition of rhTF or native hTF at 10 μg/ml, and theconcentration of viable cells was determined every day for six days.

The cell productivity of rhTF was assayed by quantifying the amount ofantibody produced in hybridoma cells at day 6 through ELISA. After cellsand debris were removed from the media by centrifugation, the antibodyquantity was measured using by ELISA as instructed by the manufacturer(Bethyl Labs, Montgomery, Tex.).

Effect of rhTF on Cell Growth and Antibody Production

Rice-derived pis-rhTF was shown to have an equivalent dose response asnative holo-hTF for the proliferation of hybridoma cells (FIG. 11A showsviable cell concentration of hybridoma cells after three days inserum-free media supplemented with no hTF, 0.03, 0.1, 0.3, 1, 5 or 30μg/ml native hTF (holo form), rice-derived rhTF, ITSE or 10% FBS). Lessthan saturating levels of activity were observed at concentrations from0.03 to 1 μg/ml with similar EC₅₀ value of about 0.3 μg/ml. Likewise, asimilar maximum effect was observed at 5 and 30 μg/ml that supportedcell proliferation to 12.0×10⁵ cells/ml. The maximum effect was similarto the ITSE cocktail control containing 5.5 μg/ml native hTF. Inaddition, hybridoma cells grown in medium with either rice-derived rhTFor native hTF showed similar growth curves (FIG. 11B shows 6 day growthcurve of Sp2/0 hybridoma in serum-free medium with either 10 μg/mlnative hTF or rice-derived rhTF, or unsupplemented), supporting thatrhTF has the same proliferation effect as native hTF. Similar effects ofrhTF and native hTF on production of antibody were also seen (FIG. 11Cshows increase in antibody production by hybridoma cells in serum-freemedium supplied with TF). These data show that pis-rhTF is equivalent tothe native holo-form of hTF in stimulating cell growth and antibodyproduction. Likely, the pis-partially iron-saturated rhTF quicklybecomes iron saturated due to the presence of iron in the medium.

While various specific embodiments have been illustrated and describedin some detail for purposes of clarity of understanding, it will beappreciated by those of ordinary skill in the art in light of theseteaching that various changes can be made without departing from thespirit and scope of the claims. Therefore, it is to be understood thatthe disclosure is not to be limited to the specific embodimentsdisclosed herein, as such are presented by way of example. It will alsobe apparent to those of ordinary skill in the art that each of theindividual embodiments described and illustrated herein has discretecomponents and features which may be readily separated from or combinedwith the features of any of the other several embodiments withoutdeparting from the scope and spirit of the teachings. Although specificterms are employed herein, they are used in a generic and descriptivesense only and not for purposes of limitation.

All literature and similar materials cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, internet web pages and other publications cited in thepresent disclosure, regardless of the format of such literature andsimilar materials, are expressly incorporated by reference in theirentirety for any purpose to the same extent as if each were individuallyindicated to be incorporated by reference. In the event that one or moreof the incorporated literature and similar materials differs from orcontradicts the present disclosure, including, but not limited todefined terms, term usage, described techniques, or the like, thepresent disclosure controls.

What is claimed is:
 1. A pharmaceutical composition comprising: a fusionprotein comprising non-glycosylated transferrin and a therapeuticprotein, wherein the fusion protein is expressed in and purified from amonocot seed.
 2. A cell culture media composition comprising: a fusionprotein comprising non-glycosylated transferrin and a therapeuticprotein wherein the fusion protein is expressed in and purified from amonocot seed.
 3. A transgenic monocot seed-derived compositioncomprising: a fusion protein comprising non-glycosylated transferrin anda therapeutic protein wherein the fusion protein is expressed in andpurified from a monocot seed.
 4. A food composition comprising: a fusionprotein comprising non-glycosylated transferrin and a therapeuticprotein wherein the fusion protein is expressed in and purified from amonocot seed.
 5. The composition of any of claims 1-4, formulated forinjectable delivery.
 6. The composition of any of claims 1-4, formulatedfor oral delivery.
 7. The composition of any of claims 1-4, wherein thetherapeutic protein is insulin or proinsulin.
 8. A transgenic monocotseed-derived composition, selected from the group consisting of awhole-seed food composition, a flour composition, an extract compositionand a malt composition, comprising a fusion protein comprisingnon-glycosylated transferrin and a therapeutic protein.
 9. Theseed-derived composition of claim 8, wherein the fusion proteincomprising non-glycosylated transferrin and a therapeutic proteinconstitutes at least 0.1% of the dry weight of the seed-derivedcomposition.
 10. The composition of claim 8 or claim 9, wherein thetherapeutic protein is insulin or proinsulin.
 11. A serum-free cellculture medium comprising an extract of transgenic monocot seedcomprising a seed-expressed fusion protein comprising: a seed-specificsignal sequence capable of targeting a polypeptide linked thereto to aprotein storage body; non-glycosylated transferrin; and a therapeuticprotein.
 12. The serum-free cell culture medium of claim 11, wherein thetherapeutic protein is insulin or proinsulin.
 13. A method of producinga recombinant fusion protein comprising transferrin and a therapeuticprotein in monocot plant seeds, comprising the steps of: (a)transforming a monocot plant cell with a chimeric gene comprising (i) aseed maturation-specific promoter; (ii) a first DNA sequence, operablylinked to said promoter, said first DNA sequence encoding a signalsequence targeting a polypeptide linked thereto to a protein storagebody of a monocot plant seed cell; and (iii) a second DNA sequence,linked in translation frame with the first DNA sequence, encoding afusion protein comprising transferrin and a therapeutic protein, whereinthe first DNA sequence and the second DNA sequence together encode afusion protein comprising the signal sequence, a therapeutic protein andtransferrin; (b) growing the monocot plant from the transformed monocotplant cell for a time sufficient to produce seeds containing the fusionprotein; and (c) harvesting the seeds from the plant.
 14. The method ofclaim 13, wherein the first DNA sequence encoding a signal sequencetargeting a polypeptide linked thereto to a protein storage body of amonocot plant seed cell encodes a glutelin signal sequence.
 15. Themethod of claim 13, wherein the fusion protein constitutes at least0.01% seed weight of the harvested seeds.
 16. A transformed rice plantproduced according to the method of claim
 13. 17. A transformed riceseed produced according to the method of claim 13.